For the past decade, this project has focused on the physiology of reactive oxygen species (ROS) and nitric oxide (NO) derivatives in respiratory and limb skeletal muscle. We have shown that these molecular cascades affect contractile function, modulating force in unfatigued muscle and contributing to fatigue during strenuous contraction. In the absence of disease, ROS and NO have also been shown to influence other aspects of cellular function in skeletal muscle. These include glucose uptake, metabolic regulation, and transcriptional control of muscle adaptation. Despite growing recognition of their physiological importance, the factors that regulate ROS and NO levels within skeletal muscle cells remain poorly understood. Speculation persists about the factors that affect cytosolic ROS and NO levels, the intracellular sites of production, and the signaling pathways that regulate ROS and NO homeostasis. The current project is designed to address these issues by evaluating redox homeostasis in skeletal muscle cells. We propose to evaluate cellular regulation of ROS and NO by diaphragm muscle fibers. A panel of established techniques will be used to detect ROS and NO in the cytosol and extracellular space. Cause-and-effect will be tested by disrupting ROS or NO signaling via pharmacologic probes and genetic engineering. We will address three Specific Aims: Aim 1. To evaluate endogenous ROS and NO activities in resting and mechanically-loaded diaphragm fibers. Aim 2. To determine the molecular source(s) and distribution of muscle-derived NO. Aim3. To evaluate principal source(s) of intracellular ROS and mechanisms that regulate ROS activity.